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OECD Test Guideline 420: Acute Oral Toxicity - Fixed Dose
- 1. Acute Oral Toxicity– Fixed Dose Procedure By Prashant Shivgunde 1/12/2015PPS, UDIRT, MUHS1
- 2. Contents 1/12/2015PPS, UDIRT, MUHS2 Introduction Initial considerations Principle of the test Description of the method Procedure Sighting study Main study Limit test Observations Data and reporting
- 3. INTRODUCTION 1/12/2015PPS, UDIRT, MUHS3 OECD Guidelines – Periodically reviewed Guideline 420 was adopted in July 1992 as the first alternative to the conventional acute toxicity test Revision was considered timely- Differ in harmonised LD50 cut-off values testing in one sex (usually females) is now considered sufficient
- 4. INTRODUCTION 1/12/2015PPS, UDIRT, MUHS4 Traditional methods for assessing acute toxicity use death of animals as an endpoint In 1984, BritishToxicology Society suggested new approach- Approach avoided using death of animals as an endpoint, and relied instead on the observation of clear signs of toxicity at one of a series of fixed dose levels. Statistical properties of the Fixed Dose Procedure have been evaluated using mathematical models in a series of studies
- 5. INTRODUCTION 1/12/2015PPS, UDIRT, MUHS5 Found way which- Uses fewer animals Causes less Suffering Is reproducible Able to rank substances in a similar manner
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- 9. Initial Considerations 1/12/2015PPS, UDIRT,MUHS9 Use of only moderately toxic doses Doses that are known to cause marked pain and distress, due to corrosive or severely irritant actions, need not be administered Moribund animals, or animals obviously in pain or showing signs of severe and enduring distress shall be humanely killed (considered in the interpretation)
- 10. Initial Considerations 1/12/2015PPS, UDIRT,MUHS10 Consider all available information on the test substance prior to conducting the study Identity and chemical structure of the substance; Its physico-chemical properties; The results of any other in vitro or in vivo toxicity tests on the substance; Toxicological data on structurally related substances; and the anticipated use(s) of the substance Helps to rank and classify according to the Globally Harmonised System (GHS)
- 11. PRINCIPLE OF THETEST 1/12/2015PPS, UDIRT, MUHS11 Groups of animals of a single sex are dosed in a stepwise procedure using the fixed doses of 5, 50, 300 and 2000 mg/kg (exceptionally an additional fixed dose of 5000 mg/kg may be considered) The initial dose level is selected on the basis of a sighting study Clinical signs and conditions associated with pain, suffering, and impending death
- 12. PRINCIPLE OF THETEST 1/12/2015PPS, UDIRT, MUHS12 Groups of animals may be dosed at higher or lower fixed doses, Depending on the presence or absence of signs of toxicity or mortality Procedure continues until the dose causing evident toxicity or no more than one death is identified, or when no effects are seen at the highest dose or when deaths occur at the lowest dose.
- 13. DESCRIPTION OF THEMETHOD 1/12/2015PPS, UDIRT, MUHS13
- 14. Selection of animalspecies 1/12/2015PPS, UDIRT, MUHS14 Rat is preferred rodent species Normally females For Males – Adequate Justification Healthy young adult animals of commonly used laboratory strains should be employed Females should be nulliparous and non-pregnant At the commencement of its dosing, should be between 8 and 12 weeks old weight should fall in an interval within + 20 % of the mean weight
- 15. Housing and feedingconditions 1/12/2015PPS, UDIRT, MUHS15 Temperature= 22ºC (+ 3ºC) & relative humidity should be 50-60% Lighting should be artificial Feeding= conventional laboratory diets with an unlimited supply of drinking water Group Caging (but No. should not interfere with clear observations of each animal)
- 16. Preparation of animals 1/12/2015PPS,UDIRT, MUHS16 Randomly selected Marked to permit individual identification, and Kept in their cages for at least 5 days prior to the start of dosing
- 17. Preparation of doses 1/12/2015PPS,UDIRT, MUHS17 Test substances should be administered in a constant volume over the range of doses In case of a liquid end product or mixture is to be tested – use undiluted test substance i.e. at a constant concentration In either case, the maximum dose volume for administration must not be exceeded In rodents maximum vol. 1 ml/100g of B.W. For Aqueous solutions – 2 ml/100g of B.W.
- 18. Preparation of doses 1/12/2015PPS,UDIRT, MUHS18 Order of administration – Liquid Suspension Emulsion Vehicles other than water? Preparation prior to administration
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- 20. Administration of doses 1/12/2015PPS,UDIRT, MUHS20 By gavage using a stomach tube or a suitable intubation canula (unusual circumstance- fraction of doses) Fasted prior to dosing Weigh animals and administer the test substance Withheld food for a further 3-4 hours in rats or 1-2 hours in mice (in case of fraction of doses?)
- 21. Sighting study 1/12/2015PPS, UDIRT,MUHS21 The purpose of the sighting study is to allow selection of the appropriate starting dose for the main study. Test substance is administered to single animals in a sequential manner following the flow charts The starting dose for the sighting study is selected from the fixed dose levels of 5, 50, 300 and 2000 mg/kg as a dose expected to produce evident toxicity
- 22. Sighting study 1/12/2015PPS, UDIRT,MUHS22 Also, evidence from in vivo and in vitro data from the same chemical and from structurally related chemicals. In the absence of such information, the starting dose will be 300 mg/kg At least 24 hours will be allowed between the dosing of each animal Consideration of use of an additional upper fixed dose level of 5000 mg/kg?
- 23. Sighting study 1/12/2015PPS, UDIRT,MUHS23 In cases where an animal tested at the lowest fixed dose level (5mg/kg) in the sighting study dies, the normal procedure is to terminate the study and assign the substance to GHS Category 1 For further confirmation use supplementary procedure
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- 27. Numbers of animalsand dose levels 1/12/2015PPS, UDIRT, MUHS27 Select dose from sighting study Perform study on a total of five animals of one sex at each level including animals tested in sighting study Time interval between dosing at each level depends on- Onset, duration, and severity of toxic signs Treatment of animals at the next dose should be delayed until one is confident of survival of the previously dosed animals
- 28. Numbers of animalsand dose levels 1/12/2015PPS, UDIRT, MUHS28 To check delayedToxicity- A period of 3 or 4 days between dosing at each dose level is recommended In case of inconclusive response Time interval may be adjusted as appropriate Fixed dose of 5000 mg/kg- procedure outlined in Annex 4
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- 31. Limit test 1/12/2015PPS, UDIRT,MUHS31 Performed when information indicating that the test material is likely to be nontoxic i.e., having toxicity only above regulatory limit doses How information about the toxicity of the test material can be gained? Using the normal procedure test will be done.
- 32. OBSERVATIONS 1/12/2015PPS, UDIRT, MUHS32 At least once during the first 30 minutes Periodically during the first 24 hours, with special attention given during the first 4 hours Daily thereafter, for a total of 14 days, Except? However, the duration of observation is not rigid Times at which signs of toxicity appear and disappear are important, especially if there is a tendency for toxic signs to be delayed
- 33. Observations 1/12/2015PPS, UDIRT, MUHS33 Should include changes in skin and fur, eyes and mucous membranes, and respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour pattern. Attention should be directed to tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma
- 34. Body Weight 1/12/2015PPS, UDIRT,MUHS34 Before Administration Thereafter weekly At end
- 35. Pathology 1/12/2015PPS, UDIRT, MUHS35 ………………should be subjected to gross necropsy.
- 36. DATA AND REPORTING 1/12/2015PPS,UDIRT, MUHS36
- 37. DATA 1/12/2015PPS, UDIRT, MUHS37 Should include Individual Animal Summarised in tabular form, Showing For each test group the number of animals used, The number of animals displaying signs of toxicity, The number of animals found dead during the test or killed for humane reasons, Time of death of individual animals, A description and the time course of toxic effects and reversibility, and Necropsy findings
- 38. Test report 1/12/2015PPS, UDIRT,MUHS38 Test substance: Physical nature, purity, and, where relevant, physico-chemical properties (including isomerisation); identification data, including CAS number. Vehicle (if appropriate): justification for choice of vehicle, if other than water. Test animals: Species/Strain used; Microbiological status of the animals, when known; Number, age and sex of animals (including, where appropriate, a rationale for use of males instead of females); Source, housing conditions, diet etc
- 39. Test report 1/12/2015PPS, UDIRT,MUHS39 Test conditions: Details of test substance formulation, including details of the physical form of the material administered; Details of the administration of the test substance including dosing volumes and time of dosing; Details of food and water quality (including diet type/source, water source); The rationale for the selection of the starting dose
- 40. Test report 1/12/2015PPS, UDIRT,MUHS40 Results: Tabulation of response data and dose level for each animal (i.e. animals showing signs of toxicity including mortality, nature, severity and duration of effects); Tabulation of body weight and body weight changes; Individual weights of animals at the day of dosing, in weekly intervals thereafter, and at time of death or sacrifice; Date and time of death if prior to scheduled sacrifice; Time course of onset of signs of toxicity and whether these were reversible for each animal; Necropsy findings and histopathological findings for each animal, if available
- 41. Test report 1/12/2015PPS, UDIRT,MUHS41 Discussion and interpretation of results. Conclusions.
- 42. Exceptional Testing Dose >2000 mg/kg 1/12/2015PPS, UDIRT, MUHS42
- 43. Dose > 2000mg/kg 1/12/2015PPS, UDIRT, MUHS43 If reliable evidence is already available that indicates the LD50 to be in the range of Category 5 values; Other animal studies or toxic effects in humans indicate a concern for human health of an acute nature Based on mortality incidences while testing schemes of Annex 3 Assignment to a more hazardous category is not warranted
- 44. Dose > 2000mg/kg 1/12/2015PPS, UDIRT, MUHS44 Reliable information is available indicating significant toxic effects in humans, Any mortality is observed when tested up to category 4 values by the oral route, Where expert judgement confirms significant clinical signs of toxicity, when tested up to Category 4 values, except for diarrhoea, piloerection or an ungroomed appearance, Where expert judgement confirms reliable information indicating the potential for significant acute effects from the other animal studies
- 45. TESTING AT DOSESABOVE 2000 MG/KG 1/12/2015PPS, UDIRT, MUHS45
- 46. Sighting Study 1/12/2015PPS, UDIRT,MUHS46 Annex 2 are extended to include a 5000 mg/kg dose level 5000 mg/kg A Second animal to be tested at 2000 mg/kg B Selection of 5000 mg/kg as the main study starting dose C Selection of 5000 mg/kg as the main study starting dose
- 47. Main Study 1/12/2015PPS, UDIRT,MUHS47 Sequential procedure presented in Annex 3 are extended to include a 5000 mg/kg dose level Main Study with 5000 mg/kg A (≥ 2 deaths) Testing of a second group at 2000 mg/kg; B (evident toxicity and/or ≤ 1 death) Substance being unclassified according to GHS. C (no toxicity) Substance being unclassified according to GHS.
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